The ability of xenoantigens to elicit the immune response represents the critical barrier in the generation of scaffolds from xenogeneic tissues for tissue engineering and regenerative medicine applications (Platt, et al., Circulation. (2002) 106:1043-1047). Decellularization approaches were originally developed with the intention of addressing antigens in xenogeneic tissues. The decellularization paradigm attributes xenograft antigenicity to the cellular component of a tissue and uses the absence of cells by light microscopy as the principle determinant of success of the process. Implantation of decellularized porcine valve tissue into sheep, rats, and dogs showed little immunogenic response for up to one year, encouraging confidence in decellularization methods (Goldstein, et al., Ann. Thorac. Surg. (2000) 70:1962-1969; Iwai, et al., J. Artificial Organs. (2007) 10:29-35). Unfortunately, in vivo studies have reported the rapid failure of SynerGraft decellularized porcine heart valves following implantation into juvenile patients (Simon, et al, Eur. J. Cardiothorac. Surg. (2003) 23:1002-1006). Failure of the acellular SynerGraft prosthetic has been attributed to inadequate xenoantigen removal with decellularization (Simon, et al., supra). Foreign body type reaction and inflammatory cell infiltration into implanted SynerGraft valves has also been demonstrated (Simon, et al., supra; Sayk, et al., Ann. Thorac. Surg. (2005) 79:1755-1758). Additionally, persistent cellular debris following the SynerGraft decellularization process has been shown to be sufficient to elicit an immunogenic reaction and calcification (Schmidt, et al., Biomaterials. (2000) 21:2215-2231). Recent studies have indicated that acellularity on light microscopy does not equate to removal of known xenoantigens from the biomaterial (Goncalves, et al., J. Heart Valve Dis. (2005) 14:212-217; Meyer, et al., J. Biomed. Mater. Res. A. (2006) 79A:254-262; Wong, et al., Acta Biomaterialia (2013) epub ahead of print (doi: 10.1016/j.actbio.2012.12.034). We have shown the lack of correlation between residual nuclei counts in bovine pericardium (BP) and residual water-soluble protein (WSP) antigenicity of the biomaterial (Wong, et al., Biomaterials (2011) 32:8129-8138). Taken together, these results indicate that the fundamental principles behind the use of decellularization as the sole process necessary for xenogeneic scaffold generation and principal determinant of biomaterial antigenicity appear to be flawed. Thus, a void remains in the development of an antigen removal (AR) process to effectively reduce xenogeneic scaffold antigenicity.
A critical error in previous decellularization approaches was focusing merely on cell disruption without regard to the need for the antigenic molecules to be solubilized for efficient removal from the xenogeneic tissue. We have demonstrated previously that by promoting the solubilization of WSPs using a reducing agent and salt to prevent intermolecular aggregation and subsequent precipitation from solution, removal of WSP antigens from BP is significantly enhanced (Wong, et al., Biomaterials, supra). Our solubilization-based AR approach reduced the residual WSP antigenicity of BP by an additional 80% compared to hypotonic solution and 60% compared to 0.1% (w/v) sodium dodecyl sulfate (SDS) decellularization methods while maintaining biomaterial tensile properties and extracellular matrix (ECM) structure and composition (Wong, et al., Biomaterials, supra). However, by only promoting WSP solubilization for removal, lipid-soluble protein (LSP) antigens are likely to persist within the tissue. Thus, a means to encourage LSP solubilization for subsequent removal, following initial WSP solubilization, could reduce overall residual antigenicity in BP post-AR (BP-AR).
The concept of differential protein solubility has long been recognized in proteomics wherein a sequential, differential approach is used for the serial extraction of protein fractions from a homogenized tissue. Protein extraction methods exploit the physiochemical properties of proteins in order to differentially and sequentially extract various subsets of proteins for downstream analyses (Beers, et al., Am. J. Physiol. (1992) 262:L773-778; DuPont. J. Agric. Food Chem. (2005) 53:1575-1584; Cordwell, et al., Methods Mol. Biol. (2008) 424:139-146; Wilson, et al., Matrix Biol. (2008) 27:709-712). The use of a series of solutions to promote protein solubilization along a spectrum of solubilities (e.g., WSP extraction followed by LSP extraction) is critical for such sequential, differential extraction protocols, since proteins can only be extracted from the material into solutions in which they are soluble (Cordwell, et al., Methods Mol. Biol., supra). However, the importance of promoting sequential, differential protein solubility during AR from intact tissues in the generation of xenogeneic scaffolds has not been investigated. This is a surprising oversight given the complex composition of protein antigens within a tissue requiring removal prior to implementation in tissue engineering applications.
The present invention is based, in part, on the discovery that a series of solutions, each promoting the solubilization and subsequent removal of a different subset of tissue proteins based on their solubility, enhances overall AR from BP. Furthermore, such a sequential, differential AR strategy significantly reduces BP antigenicity while maintaining biomaterial functional properties. In this study, several LSP solubilization promoting agents were applied as a second step of AR following initial WSP solubilization and assessed for their ability to reduce the residual LSP antigenicity of the resultant BP-AR. The effectiveness of this two-step sequential, differential strategy for reducing LSP antigens in BP was compared to a one-step AR strategy (WSP solubilization) (Wong, et al., Biomaterials, supra) and the literature gold standard (1% (w/v) SDS). See, Wong, et al., Acta Biomaterialia (2013) epub ahead of print (doi: 10.1016/j.actbio.2012.12.034). The effect of this two-step AR protocol on ECM mechanical properties, structure and composition of BP-AR was assessed by uniaxial tensile testing, histological analysis and biochemical quantification of ECM components, respectively.
Previously described myocardial tissue decellularization methods have also been solely based on the ubiquitous use of harsh denaturing detergents, mainly SDS, in concentrations as high as 2% in hypotonic water solutions. See, Elder, et al., Biomaterials. (2009) 30(22): 3749-3756. Although effective in solubilizing cellular and tissue components, it has been shown that these methods are not successful in removing antigenic determinants, while they are often detrimental to the extracellular matrix (removing elastin, glycosaminoglycans and damaging collagen structure). Further, commonly utilized detergents are toxic to repopulating cells reducing the chances of successful recellularization strategies for the produced scaffolds.
Reported methods to produce a myocardial scaffold have been based on detergent-based decellularization methods. More specifically, these approaches included the use of one detergent (SDS, Triton-X100, Saponin) with protease inhibitors (to prevent extracellular matrix protein degradation) and occasionally enzymatic treatments (such as trypsin) and nucleases for nucleic acid degradation in different concentrations and combinations. All these reports determine loss of nuclei and cellular components and production of an acellular scaffold as their outcome measure for protocol success. As we have already shown in our laboratory this assumption is not valid, since antigens may be associated with non-cellular components of the tissue. Additionally, the omission of a reducing agent in all these treatments would be expected to result in protein precipitation rather than solubilization and extraction, regardless of the concentration of the detergent used.